Processes to prepare elongated 2-ketoacids and C5-C10 compounds therefrom via genetic modifications to microbial metabolic pathways

Abstract

Genetically modified isopropylmalate synthases, processes for preparing a C7-C11 2-ketoacids utilizing genetically modified isopropylmalate synthases, and microbial organisms including genetically modified isopropylmalate synthases are described. The genetically modified isopropylmalate synthases, processes for preparing a C7-C11 2-ketoacids, and microbial organisms including genetically modified isopropylmalate synthases can be particularly useful for producing corresponding Cn_1 aldehydes, alcohols, carboxylic acids, and Cn_2 alkanes both in vivo and in vitro.

What is claimed is: 
     
       1. A process for preparing a C 7 -C 11  2-ketoacid, the process comprising: (I) providing at least one of a C 4 -C 10  2-ketoacid substrate, with:
 (a) a genetically modified isopropylmalate synthase (IPMS) comprising at least one of: 
 (i) an isopropylmalate synthase variant comprising the amino acid sequence variant of SEQ ID NO: 1 having mutations H97A, S139G, N167G, P169A, G181A, A182G, G210A, A214S, and G462D in the SEQ ID NO: 1 and having IPMS activity; (ii) isopropylmalate synthase variant comprising the amino acid sequence variant comprising SEQ ID NO: 2 having the mutations H97A, S139G, N167G, P169A, M255L, R260A, N264Q, and G462D in the SEQ ID NO: 2 and having IPMS activity; or (iii) isopropylmalate synthase variant comprising the amino acid sequence variant of comprising SEQ ID NO: 2 having the mutations H97A, S139G, N167G, P169A, D348E, D350E, M353L, Q355N, and G462D in the SEQ ID NO: 2 and having IPMS activity 
 (b) a isopropylmalate isomerase having isopropylmalate isomerase activity; and 
 (c) a isopropylmalate dehydrogenase having isopropylmalate dehydrogenase activity; 
 under conditions that the at least one of the C 4 -C 10  2-ketoacid substrate is converted to the C 7 -C 11  2-ketoacid; and wherein the conversion of the at least one of the C 4 -C 10  2-ketoacid substrate to the C 7 -C 11  2-ketoacid occurs via one or more biochemical reactions. 
 
     
     
       2. The process according to  claim 1 , wherein the at least one of the C 4 -C 10  2-ketoacid substrate comprises 2-ketobutyrate. 
     
     
       3. The process according to  claim 1 , wherein the at least one of the C 4 -C 10  2-ketoacid substrate comprises 2-ketoisovalerate. 
     
     
       4. A genetically modified isopropylmalate synthase (IPMS) polypeptide having isopropylmalate synthase activity, the polypeptide comprising at least one of: (a) an isopropylmalate synthase variant comprising the amino acid sequence variant of SEQ ID NO: 1 having mutations H97A, S139G, N167G, P169A, G181A, A182G, G210A, A214S, and G462D in the SEQ ID NO: 1 and having IPMS activity; (b) isopropylmalate synthase variant comprising the amino acid sequence variant comprising SEQ ID NO: 2 having the mutations H97A, S139G, N167G, P169A, M255L, R260A, N264Q, and G462D in the SEQ ID NO: 2 and having IPMS activity; or (c) isopropylmalate synthase variant comprising the amino acid sequence variant of comprising SEQ ID NO: 2 having the mutations H97A, S139G, N167G, P169A, D348E, D350E, M353L, Q355N, and G462D in the SEQ ID NO: 2 and having IPMS activity. 
     
     
       5. The genetically modified isopropylmalate synthase polypeptide having isopropylmalate synthase activity of  claim 4 , wherein the polypeptide comprises the isopropylmalate synthase variant comprising the amino acid sequence variant of SEQ ID NO: 1 having mutations H97A, S139G, N167G, P169A, G181A, A182G, G210A, A214S, and G462D in the SEQ ID NO: 1 and having IPMS activity. 
     
     
       6. The genetically modified isopropylmalate synthase polypeptide having isopropylmalate synthase activity of  claim 4 , wherein the polypeptide comprises the isopropylmalate synthase variant comprising the amino acid sequence variant comprising SEQ ID NO: 2 having the mutations H97A, S139G, N167G, P169A, M255L, R260A, N264Q, and G462D in the SEQ ID NO: 2 and having IPMS activity. 
     
     
       7. The genetically modified isopropylmalate synthase polypeptide having isopropylmalate synthase activity of  claim 4 , wherein the polypeptide comprises the isopropylmalate synthase variant comprising the amino acid sequence variant of comprising SEQ ID NO: 2 having the mutations H97A, S139G, N167G, P169A, D348E, D350E, M353L, Q355N, and G462D in the SEQ ID NO: 2 and having IPMS activity.